Variants of a transcription factor (TF) binding motif among genes are commonly thought to be functionally equivalent (degenerate), but some of them may in fact influence gene expression;such a variant is called a "functional" variant. Little is known about how or when these functional variants act, and how to detect them. Our goal is to understand the extent that TF binding site variants contribute to expression variation among genes in a genome or between genes in different species. Our aims are to: A. Characterize functional TF binding site variants in yeast using computational approaches, (a) Develop robust methods to account for multiple binding sites of the TF, degeneracy within functional variants, expression noise, and non-independence of all-by-all comparisons of genes and binding site variants, (b) Elucidate the patterns of conservation of variant-specific expression profiles, looking across Saccharomyces sensu stricto species and between these species and C. albicans to determine whether the function of target genes of TFs or the sequence composition of the motif is associated with the conservation of functional variants, (c) Characterize nucleotide substitutions in target gene promoters that may have caused a switch in variant-specific expression profiles between species B. Experimentally validate and characterize functional variants, (a) Alter nucleotides at variant binding site positions using site-directed mutagenesis to test for predicted expression changes. A number of variant sites in S. cerevisiae will be selected for experimental validation. Further, the effect of variant switches between S. cerevisiae and S. paradoxus will be validated by allele-specific expression analysis in a hybrid strain, (b) Test models for the mechanism of variant binding site function. The proposed research will increase our understanding of the complex cis-regulatory code that underlies physiology and pathology. As mutations in promoters have been associated with heart disease, HIV transmission risk, alpha-thalassemia, Alzheimer's and other diseases, understanding the full range of function of a binding site could be critical for assessing mutations that are associated with disease etiology.